Alternative pre-mRNA splicing, a process generating multiple distinct mRNA transcripts from a single precursor transcript, has played a critical role in the evolution of transcriptome complexity and cellular diversification in metazoans. It is therefore not surprising that alternative splicing events are frequently subject to regulatory control in a cell- and tissue-type dependent manner. However, despite significant advances made in our understanding of the mechanisms governing tissue-specific alternative splicing regulation, there is still much to learn. For instance, only a small number of splicing factors with tissue-specific expression patterns have been identified, and collectively these factors cannot account for the degree of alternative splicing regulatory complexity observed in diverse cell-types. Even less is known about the extent and control of differential regulation of splice variants between individual cells of a tissue-type or organ, owing in part to limitations in the detection of these isoforms. Finally, only a few studies have begun to address the functional roles of tissue-specific splicing regulators and the networks of splicing events they control in cellular differentiation and specialization. Here, we propose to use the Caenorhabditis elegans nervous system as a model to advance our understanding of alternative splicing regulation in vivo. First, we will employ genome- wide approaches to elucidate the regulatory network controlled by the neuron-specific CELF family splicing protein UNC-75, a factor known to be required for proper neuronal function. We will then perform a detailed investigation of this network using genetic techniques and isoform-specific analyses to identify genes and splice variants that are the most significant downstream effectors of UNC-75 in modulating neuronal function. Finally, we propose to conduct genetic screens to identify novel regulators of neuronal cell-subtype specific alternative splicing patterns. The identification of these cell-subtype specific regulators will open the door to additional investigation of alternative splicing networks in specific classes of neurons. Taken together, the aims described in this application will reveal new insights into how regulation of alternative splicing at the level of individual cells can be achieved, and how the study of alternative splicing regulatory networks can be used to infer previously unknown functions of genes and isoforms in key developmental processes. PUBLIC HEALTH RELEVANCE: The goal of this application is to determine how cells of the nervous system can interpret their genetic material to produce the building blocks necessary for key functions such as the transmission of electrical signals and interpretation of environmental cues. State-of-the-art approaches will be used to make fundamental discoveries about how these basic processes influence the health and diverse abilities of organs such as the brain.